Project Summary Flagella and the synonymous cilia protrude above the cell surface to detect signals and whip surrounding fluid. These remarkable cellular antennae and propellers play central roles in human development and the operations of most organs. Yet to grow and maintain the structural core supporting these hair-like organelles, a unique train-like system termed intraflagellar transport (IFT) must continuously deliver a variety of proteins made in the cell body to the distal tip of flagella. Defects in trafficking and the structure core underlies many debilitating genetic disorders and chronic illnesses. While IFT has been investigated extensively, how and where IFT trains load various core proteins remains elusive. Founded on technical breakthroughs in the single cell protist, Chlamydomonas, which is amenable to diverse experimental approaches, preliminary data provides an unprecedented opportunity to elucidate this complicated process and involved genes. A model protein complex, the radial spoke, fails to assemble at the distal part of flagella in the mutant strains defective in the ARMC2 gene or PF5 gene. Under a microscope, fluorescent ARMC2 and IFT trains are concentrated near the flagellar entrance, whereas radial spokes are abundant and dynamic in a novel ?cargo hub? in the cell body. This proposal aims to test objectively and rigorously the hypothesis that the ARMC2-PF5 adaptor facilitates and streamlines radial spoke trafficking from the cytoplasmic cargo hub to the flagellar distal tip for assembly. Aim 1 will generate transgenic strains expressing ARMC2, radial spokes and IFT trains that are tagged to fluorescent proteins of superior optical properties. Various imaging systems and quantitative analysis will be used to reveal the locations of fluorescent molecules and to track their movements; and to determine how growing or shrinking flagella affect the protein cargoes in the putative cargo hub. Aim 1 will further test if the distal deficiency will be lessened by slowing ciliary growth rates to allow vestigial assembly to catch up. Aim 2 will use sequencing, genetic and biochemical approaches to determine if the ARMC2 gene or PF5 gene are identical or distinct; and to identify candidate ARMC2 interactors, such as radial spokes, IFT trains and perhaps PF5 protein. Aim 3 will use molecular approaches to test whether the major segments in ARMC2 are involved in binding IFT trains and radial spokes respectively. Together, the proposed projects will define the new cargo hub and reveal the molecular basis that enables IFT trains pick up and drop off cargoes at proper locations. The findings and innovative approaches will stimulate novel ideas in the ciliary field, empower researchers facing similar challenges, and accelerate the discoveries of similar atypical flagellar genes that are likely causative to cilia-related diseases. Modulation of relative assembly rates of individual complexes and the entire flagellum will encourage the development of innovative therapeutic strategies. Finally, the combination of classical experiments, cutting-edge technologies and creative problem-solving strategies will attract and prepare graduate and many undergraduate students to pursue careers in the future biomedical fields.